Diffusion through a membrane assaying apparatus and method

ABSTRACT

The diffusion through a membrane assaying apparatus and method facilitates rapid detection of small or larger molecular weight substances such as hazardous wastes, toxic chemicals or the like by using a semipermeable membrane having a predetermined molecular weight cutoff. The semipermeable membrane is provided as part of a container having a removable barrier which facilitates control of diffusion through the membrane. The assaying method includes the use of a reaction mechanism for detection of a predetermined substance. The reaction mechanism includes one or more reagents which are designed to either react or compete for a substance for which assaying is being performed. By selecting the proper reagents and molecular weight cutoff of the semipermeable membrane, the presence or absence of a reaction such as a color change or production of vapor provides indication whether the substance being assaying for is present in the test sample.

This application is a continuation of Ser. No. 08/173,854 filed Dec. 23,1993 and Ser. No. 07/928,121, filed Aug. 12, 1992, both now abandoned,and a division of Ser. No. 371,711, filed Jan. 12, 1995, now U.S. Pat.No. 5,525,475.

FIELD OF THE INVENTION

The present invention is directed to a diffusion through a membraneassaying apparatus and method, and in particular, a portable containeror a membrane in the form of a container which facilitates the rapiddetection of small or large molecular weight substances such ashazardous wastes, pesticides, toxic chemicals, chemical warfare agents,proteins from infectious organisms or the like.

BACKGROUND OF THE INVENTION

In the prior art, various methods and devices have been proposed forassaying various substances. U.S. Pat. No. 4,770,853 to Bernsteindiscloses a device for self-contained solid-phase immunodiffusion assaycomprising a sample collector, a tube with compartmentalized reagentsand a ligand receptor capture membrane filter area. The sample collectoris pushed through seals in the device and is mixed with reagent, andthen pushed into a ligand receptor reaction area. A tip of the samplecollector contacts diffusible membranes and transfers the reactants to acapture membrane for visualization of a ligand receptor reaction by aviewer.

U.S. Pat. No. 4,818,677 to Hay-Kaufman et al. discloses methods and akit for performing immunoassays. The kit includes a reaction cell havinga micro-porous membrane and an absorbent capable of drawing a liquidsample through the membrane. Analyte in the sample is immobilized,typically by immunoadsorption, and the immobilized analyte may then bevisualized using conventional signal producing systems, such as color,fluorescence and luminescent systems.

U.S. Pat. No. 5,043,260 to Jauregui discloses a perfusion device to growand maintain hepatocytes including a chamber having a perfusion inletand a perfusion outlet. A semipermeable membrane in the chamber definesseparate perfusion and hepatocyte compartments, the hepatocytes thereinbeing attached via oligosaccharide-lectin recognition linkage to abiopolymer support in the hepatocyte compartment.

The prior art also teaches immunochemically-based methodologies for thedetection of small or large molecular weight substances. Exemplary ofthese methodologies include radioimmunoassay, enzyme-linkedimmunosorbent assay, enzyme multiplied immunoassay, enzyme immunoassay,fluorescent immunoassay, nephelometric inhibition immunoassay,hemagglutination inhibition, microbiologic assay, free radical assaytechnique--also called spin immunoassay, liposome immunoassay andpotentiometric immunoassay.

U.S. Pat. Nos. 5,002,871 and 5,037,741 to Iacobucci disclose a membranemethod for the enzymatic synthesis of peptides. The method employs amembrane permeable to uncharged peptides but impermeable to chargedmolecules. The enzymatic method provides a process for the safe,economical and efficient synthesis and purification of peptides andderivatives thereof, and the efficient use of enzymes and the means toaffect the synthesis on a continuous basis.

Disadvantages of currently used immunoassays include difficulty whenusing under field conditions, expensive, difficult to perform withouttraining, time consuming and the requirement of multiple reagents, manyof which can undergo degradation and, thus, interfere with test results.In view of the deficiencies in prior art detection methodologies, a needhas developed to provide a rapid, portable, easy to use, inexpensive andversatile assaying apparatus and method.

In response to this need, the present invention provides an assayingapparatus and method which overcomes the deficiencies in the prior art.The inventive assaying apparatus and method provides results within afew minutes, requires a small number and small volume of reagents,utilizes simple chemical reactions to perform given assays, provideslong shelf life reagents and provides easily interpreted assay results.

SUMMARY OF THE INVENTION

It is accordingly a first object of the present invention to provide anassaying apparatus and method utilizing diffusion through a membrane.

It is another object of the present invention to provide an assayingapparatus that provides results in a short period of time, is portablefor field environment use, is simple to use and is inexpensive.

A further object of the present invention is to provide an assayingapparatus and method of assaying which requires a small number and smallvolume of reagents, thereby decreasing assaying unit costs.

Another object of the present invention is to provide an assayingapparatus and method which offers easily interpreted results for assaydetermination.

Other objects and advantages of the invention will be apparent as thedescription thereof proceeds.

In satisfaction of the foregoing objects and advantages, in a firstembodiment, the present invention comprises an assaying apparatus fordetecting a wide range of molecular weight substances including acontainer having first and second chambers therein. The first and secondchambers are separated by a removable non-permeable barrier and anadjacent semipermeable membrane. The semipermeable membrane is selectedhaving a molecular weight cut off value in accordance with theparticular substance for which assaying is being performed. The firstand second chambers each have an opening and a cap adapted to close eachopening. During assaying, the non-permeable barrier is removed to permitdiffusion through the semipermeable membrane and between the twochambers.

Various reagents are provided for addition to each of the chambers topermit assaying a sample for determination of a particular molecularweight substance. The chambers may be configured to facilitate visualdetection of reactions during assaying and can include caps with indiciathereon to further facilitate particular assaying methods.

In an alternative embodiment, the semipermeable membrane is provided ina container form. In this embodiment, the semipermeable membranecontainer is utilized with a reaction vessel and reagents for performingthe various assaying methods. In one embodiment, the reaction vessel maybe a closed vessel wherein the vessel cap includes provision for storingreagents. The semipermeable membrane container can also be utilized withan open reaction vessel such as a Petri dish.

The assaying apparatus can be used with a variety of reagents fordetection of both small and large molecular weight substances. In onemode of the inventive assaying method, color changes, production ofvapors or intensity of color changes or vapor production are utilized tofacilitate detection of a given substance. During the assaying methods,the molecular weight cut off value of the semipermeable membranecontrols diffusion across this membrane. Thus, and depending upon thesize of the reagents used, a user can select the reagents in conjunctionwith the semipermeable membrane molecular cut off weight size toidentify particular molecular weight substances.

In one particular mode of assaying, an antigen to be tested for islabeled with a labeling agent. The labeled antigen is associated with aparticular antibody to establish a given molecular weight substance.During assaying, and if antigens are present in a test sample, theantigens replace the labeled antigens on the antibodies. Since thelabeled antigens are small enough in size to diffuse through thesemipermeable membrane, the presence of the antigen can be detected as aresult of the labeled antigen diffusion and producing a correspondingcolor change.

In an alternate mode of assaying, an enzyme substrate system is providedwherein the enzyme acts as the labeling agent. By controlling diffusionwith the semipermeable membrane and the size of the substance which theenzyme labels, the reaction between the enzyme and substrate, either asa color change or vapor production, is controlled to indicate detectionof a given substance.

Besides selecting particular reagents for the inventive assaying method,the reagents and a sample to be tested for detection of a substancehaving a predetermined molecular weight can be combined in apredetermined sequence with the containers as described and reactionvessels to obtain an assaying result by diffusion through thesemipermeable membrane of select reagents.

BRIEF DESCRIPTION OF DRAWINGS

Reference is now made to the drawings accompanying the applicationwherein:

FIG. 1a depicts a first embodiment of the inventive assaying apparatuswith the non-permeable barrier intact;

FIG. 1b shows the apparatus depicted in FIG. 1a with the non-permeablebarrier removed, thus exposing the semipermeable membrane;

FIG. 2 shows a perspective view of a multiple chamber assayingapparatus;

FIG. 3 shows a perspective view of another configuration of the assayingapparatus depicted in FIG. 1;

FIGS. 4a-4c show the assaying apparatus of FIG. 1 in exemplary modes ofuse;

FIGS. 5a-5e are diagrammatic representations of an exemplary reactionsequence using the assaying apparatus of FIG. 1;

FIG. 5f is a key identifying substances used in the reaction sequencedepicted in FIGS. 5a-5e;

FIGS. 6a-6c show a diagrammatic representation of an exemplary reactionsequence when the absence of vapors indicates a positive reaction;

FIG. 6d illustrates a key for identifying substances in FIGS. 6a-6c andFIGS. 7a-7c;

FIGS. 7a-7c show a diagrammatic representation of an exemplary reactionsequence when the presence of vapors indicates a negative reaction;

FIG. 8 shows a second embodiment of an unassembled assaying apparatusand a key identifying exemplary reagents for use in assaying;

FIGS. 9A, 9B, 9C shows a diagrammatic representation of an exemplaryreaction sequence using the apparatus and key depicted in FIG. 8;

FIGS. 10a and 10b show another embodiment of the inventive assayingapparatus along with a diagrammatic representation of an exemplaryreagent for use in assaying;

FIGS. 11a-11d show a diagrammatic representation of a reaction sequenceutilizing the embodiment depicted in FIG. 10a;

FIG. 12 is a flowchart describing the reaction sequence steps for FIGS.11a-11d;

FIGS. 13a-13c show a diagrammatic representation of a reaction sequencewhen the absence of vapors indicates a positive reaction;

FIG. 14 is a flowchart describing the reaction sequence for FIGS.13a-13c;

FIGS. 15a-15d show another diagrammatic sequence of a reaction schemewhen the presence of greater concentrations of vapors indicates apositive reaction;

FIG. 16 is a flowchart describing the reaction sequence for FIGS.15a-15d;

FIGS. 17a-17d show another diagrammatic representation of a reactionsequence when the presence of less vapors indicates a negative reaction;and

FIG. 18 is a flowchart describing the reaction sequence steps for FIGS.17a-17d.

FIG. 19 shows a conjugate comprising antibodies bound to ferritin,microspheres, or colloidal gold.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

The inventive assaying apparatus and method utilizing diffusion througha membrane solves the prior art problem of detecting both small andlarge molecular weight substances in a single assaying apparatus. Theinventive assaying apparatus is capable of detecting small molecularweight substances such as chemical warfare agents, drugs (boththerapeutic and those of abuse), hormones such as progesterone, andtoxins, e.g., T-2 toxin. The inventive assaying apparatus and methodalso includes capability of detecting large molecular substancesincluding a variety of proteinaceous substances from infectiousorganisms, e.g., bacteria, fungi and parasitic organisms, and antibodiesdirected against these substances.

The inventive assaying apparatus and method includes a wide variety ofapplications. For example, the assaying apparatus can be used toidentify samples that contain life-threatening chemicals such as warfareagents by members of the military. Moreover, rapid detection of drugs inbiological samples such as urine can be achieved using the inventiveassaying apparatus and method. Drugs can be constantly monitored fortherapeutic use or when people or animals, e.g. race horses, have takenor have been given drugs of abuse.

The assaying apparatus and method may be used to determine hormonelevels in body fluids to monitor patients, monitor athletes and monitorhormone levels for ovulation determination in women. The capability ofdetecting toxins makes the inventive assaying apparatus useful for allbranches of the military and in medical related applications.

With reference now to FIGS. 1a and 1b, a first embodiment of thediffusion through a membrane (hereinafter DITAM) apparatus is generallydesignated by the reference numeral 10 and is seen to include a handheldtransparent container 1 with first and second chambers, 3 and 5,respectively. The container 1 can be constructed from polystyrene oranother optically clear and transparent material such as glass. Withreference to FIG. 1a, each of the chambers 3 and 5 have a cap 7 whichcovers an opening in the respective chambers. Disposed between chambers3 and 5 is a removable non-permeable barrier 9.

With reference to FIG. 1b, the non-permeable barrier 9 is removable fromthe container 1 via the slot 11 in a top portion thereof. Once thenon-permeable barrier 9 is removed, a semipermeable membrane 13 isexposed, the semipermeable membrane being positioned between chambers 3and 5. The semipermeable membrane is not removable from between chambers3 and 5.

The molecular weight cutoff (hereinafter "m.w.c.o.") of the membrane 13can be varied depending on the substance to be tested for. For example,the DITAM apparatus used to test for small molecular weight substanceswould utilize semipermeable membranes which have a relatively small poresize. Conversely, the DITAM apparatus used to test for large molecularweight substances would utilize semipermeable membranes which have arelatively large pore size. Although a variety of membranes can beemployed, dialysis-type membranes are the most useful. This is due tothe fact that molecules can easily migrate through the membrane when anindividual is gently shaking the apparatus from side to side. Othermembranes can be employed, but positive or negative pressure may berequired for the molecules to traverse the membrane. For example, theapparatus can be reconfigured for use with a vacuum system if themembrane type requires the use of a vacuum for the molecules to migratethrough the pores. Since this device was intended for field use, the useof a vacuum system with the apparatus is not desirable. Additionaldetails and examples of actual membranes used for each embodiment of theDITAM apparatus are discussed hereinafter.

The caps can be produced from a colored material. If desired, caps canbe produced in many different colors. Cap color may be used todifferentiate an apparatus that is used for a particular test. Forexample, a DITAM apparatus with an orange cap can be reserved to testfor substance "A". A DITAM apparatus with a blue cap can be reserved totest for substance "B". Caps can also be transparent and devoid ofcoloring. All of the instructions for performing a DITAM assay can beprinted on one or two sides of the apparatus.

With reference now to FIG. 2, a four-chambered apparatus is designatedby the reference numeral 20 and is seen to include a first pair ofchambers 3 and 5 separated by a first non-permeable barrier 9 and asecond pair of chambers 3' and 5' separated by a non-permeable barrier9'. In this configuration, a control sample can be tested in chambers 3and 5 with a test sample assayed in chambers 3' and 5'. As describedabove, the non-permeable barriers 9 and 9' of the four-chamberedapparatus are removed by lifting upwardly to expose the semipermeablemembranes described previously. The four-chambered apparatus 20 alsoincludes caps 7' which are also removable to permit access to thevarious chambers.

The four-chambered apparatus is shown having a length designated by "L",a depth "D" and a width "W". Exemplary dimensions for the four-chamberedapparatus 20 include a length of 44 mm, depth of 12 mm and a width of 51mm. Of course, the four or two chambered apparatus can be made withdifferent dimensions to accommodate larger or smaller reagent and samplevolume sizes. Further, a different size may also be constructed for usewith a particular mechanical optical density reading device.

When viewing a solution in chambers 3' and 5' of the four-chamberedapparatus 20, a viewer can look at the apparatus through a front view,or with the cap removed, through a top view. Given exemplary dimensionsof a 12 mm depth and a 44 mm height, viewing a reaction solution throughthe front view makes the solution appear lighter. In contrast, when thereaction solution is viewed through a top view with a path length of 44mm, the solution appears darker. The difference in path lengths allowsfor greater test sensitivity when viewing along a longer path lengthwithout the need for a mechanical reading device.

An elongated version of the apparatus disclosed in FIGS. 1a and 2 isdepicted in FIG. 3. The elongated version allows an individual to view acolored reaction solution through a longer path length, and the color ofthe reaction solution appears to be darker. Thus, it is easier for anunaided eye to visualize test results. The DITAM apparatus depicted inFIG. 3 is generally designated by the reference numeral 30 and is seento include a container 1' divided into chambers 3" and 5" bynon-permeable barrier 9". The caps 7" are disposed on a top surface ofeach of the chambers such that the test apparatus rests on a surfaceopposite a surface containing the openings for the caps 7". In theapparatus 30, exemplary dimensions include a length of 88 mm, a depth of12 mm and a width of 25 mm. Again, the apparatus 30 can be made withdifferent dimensions to accommodate larger or smaller reagent and samplevolume sizes. A different size may also be constructed for use with aparticular mechanical optical density reading device.

As set forth above, the non-permeable barrier 9" is removed by liftingupwardly to expose the semipermeable membrane positioned between chamber3" and 5".

It should be understood that the configuration, sizes and shapes of theassaying apparatus 10, 20 and 30 are merely exemplary and, as such, maybe constructed in different sizes and shapes to accommodate the needs ofa user.

In its simplest form, the DITAM assay requires the following reagents:antibody molecules which recognize a specific antigen (or severaldifferent antigens), specific antigens labeled with chromophores orfluorophores, and a buffer solution. The test sample may contain antigenor it may be devoid of antigen. The antigen is the specific substancethat is being tested for. This antigen (or several antigens) arespecifically recognized by the antibody molecules that are employed inthe assay. In its simpleest form, the DITAM assay utilizes a chromophoreor fluorophore to label the antigen and to indicate a positive ornegative test result. FIGS. 4A-4C illustrate the use of fuchsin red isas a label for antigens. Prior to testing, the solution in chamber 3 ofthe DITAM apparatus is shown as shaded (FIG. 4A), but is pink in actualuse. When a test is completed, a negative reaction (no antigen in testsample) appears shaded in chamber 3 and relatively clear in chamber 5(FIG. 4C). When a test is completed, a positive reaction (antigen intest sample) appears light pink in both chambers 3 and 5 (FIG. 4B).

Several different labeling substances may be used in place of thechromophores or fluorophores. A variety of enzymes may be used to labelthe antigens. If these are employed, an extra step is added to the test;and the appropriate enzyme-substrate solution is required. If used, thecap of the apparatus could be configured to enable the storage of theenzyme-substrate solution in the cap. The reaction of enzyme andenzyme-substrate solution may produce a colored reaction product.Alternatively, the reaction of enzyme and enzyme-substrate solution mayproduce vapors; and these vapors could be detected by a user with achemical vapor detection device.

All reagents can be supplied in lyophilized form and can bereconstituted with a buffer solution immediately prior to performing anassay. In lyophilized form, the DITAM apparatus and the assay reagentscan be stored at 4° C. or a lower temperature until ready for use.Degradation of these reagents will be halted or delayed substantially ifstored properly. A stabilizer may also be added to the chamber toprolong the shelf life of the reagents. Thus, reagents do not degrade asrapidly when exposed to warm environments.

The test sample may be a liquid, i.e., contaminated water, saliva, serumor urine. Alternatively, solid test samples, i.e., contaminated dustparticles, can be concentrated on cotton swabs and placed in test tubesalong with a buffer solution. This process applies to all of theversions of the DITAM Assay.

The direction for performing a DITAM Assay will now be described. Priorto performing a DITAM Assay, an individual must select a DITAM apparatuswhich contains a semipermeable membrane with the appropriate pore sizeor molecular weight cutoff (m.w.c.o.). This membrane is discussedhereinafter. The caps are removed and reinserted as needed to addreagents to the chambers. Lyophilized reagents are reconstituted with abuffer solution or with 0.85% saline immediately prior to use.Alternately, an individual may reconstitute the reagents in severalapparatus on the morning that the assays are to be performed. In thismanner, one step is eliminated when assays are performed in the field orin the lab. Reconstitution of reagents several days in advance is notrecommended since this may accelerate degradation of the reagents.

When an individual is ready to perform a DITAM assay, the test sample isadded to chamber 5 of the apparatus; and the nonpermeable barrier isremoved by lifting upwards. The individual then shakes the DITAMapparatus to allow smaller molecules to diffuse through thesemipermeable membrane. After 2-3 minutes, the individual observes theapparatus for color changes in both chambers. This can be accomplishedwith the unaided eye or a small spectrophotometer. For field use, theformer method of observation is preferred. Color in chamber 5 indicatesthe presence of antigen in the test sample. One type of color changethat may be observed if a chromogen such as fuchsin red is used as alabel for antigens is various shades of pink. The intensity of the coloris dependent on the amount of chromogen on each side of the membrane.

Using a four-chambered apparatus as shown in FIG. 2, the two chambers,3' and 5', can be used as a control for comparison with chambers 3 and 5to assay a test sample. In this manner, chambers 3 and 5 should indicatea negative reaction (no antigen in the test sample) or a positivereaction (antigen in the test sample) with the two chambers, 3' and 5',illustrating a positive reaction or antigen present in the controlsample. Although a chromogen such as fuchsin red has been describedabove for indicating the presence of an antigen, other indicating agentssuch as fluorescein may be used as a label for antigens. Usingfluorescein, the reaction solutions appear as various shades of yellow.Again, when viewing for color changes using the assaying apparatus,viewing along a top view gives the appearance of a darker solution whichis easier to visualize with an unaided eye.

With reference now to FIGS. 5A-5F, a diagrammatic representation of anexemplary reaction sequence for the DITAM assay will now be described.Once the correct DITAM apparatus is selected, the reactions can proceed.Lyophilized antigen-chromogen (chromophore) conjugates bound to antibodymolecules or antigen-fluorophore conjugates bound to antibody molecules21 are placed in chamber 3 of the apparatus, (FIG. 5A). Reconstitutionis accomplished by adding a buffer solution or 0.85% saline to chamber3, and the molecules go into the solution (FIG. 5B). The solutionbecomes colored due to the presence of the chromophore or fluorophore.Many different colored solutions are possible depending on the color ofthe chromophore or fluorophore. For example, fuchsin red produces a pinkcolored solution. The intensity of the color is directly dependent onthe concentration of fuchsin red in the solution. Fluorescein produces ayellow colored solution; and again, the intensity of the color isdirectly dependent on the concentration of fluorescein in the solution.Ideally, for field testing, it is desirable to utilize a sufficientquantity of the chromophore or fluorophore to enable the visualizationof the colored solution with the unaided eye. If this not possible, ahand-held spectrophotometer may be required to detect the color changesduring the course of the reactions.

The test sample is added to chamber 5 (FIG. 5C). A liquid sample may betested, and this sample may or may not contain the antigen to be testedfor. In the illustration, the antigen 23 is present in the test sample.If a solid test sample is added to chamber 5 of the apparatus, then abuffer solution or 0.85% saline must be added to chamber 5 in order tohave the test sample go into solution. The nonpermeable barrier isremoved by lifting upwards. This barrier may be removed completely; orit may be elevated to the level of the caps so that the barrier can bereinserted at a later time.

Once the nonpermeable barrier is removed, the permanently affixedsemipermeable membrane is exposed, (FIG. 5D). The semipermeable membranemust have a m.w.c.o. that prohibits the antibody molecule from crossingfrom chamber 3 to chamber 5. The membrane must also have a m.w.c.o. thatenables the antigen from the test sample and the antigen-chromophore orantigen-fluorophore conjugate to migrate multidirectionally betweenchamber 3 and 5 in a few minutes time. If antibody molecules of the IgGclass are employed (m.w. approximately 150,000), then the m.w.c.o. ofthe membrane must be smaller than 150,000. In order to ensure that theantibody molecules do not migrate through, a membrane with a m.w.c.o. of50,000 or 100,000 should be selected. When testing for small molecularweight substance, i.e., in the range of the hundreds or low thousands,this membrane would easily allow these smaller molecules to migratemultidirectionally through the membrane. When the individual shakes theapparatus, the smaller molecules migrate through the semipermeablemembrane. The concentration of these small molecules on each side of themembrane is ultimately dependent on osmotic equilibrium. However, duringthe short duration of the assay, equilibrium is generally not reached.Once antigen molecules from the test sample pass from chamber 5 tochamber 3, another reaction occurs. Some of the antigen-chromophore (orantigen-fluorophore) conjugates become disassociated from the antibodymolecules. Once one or two of the binding sites are exposed each of theantibody molecules, antigen from the test sample can bind to theantibody molecules. With greater concentrations of antigen molecules inthe test sample, more will pass through the membrane; and more of theseantigen molecules will displace the antigen-chromogen (orantigen-fluorophore) conjugates from the antibody molecules. Afterdisplacement occurs, the antigen-chromogen (or antigen-fluorophore)conjugates will migrate through the membrane. The end result of thismigration is that chromophores (or fluorophores) will be present on bothsides of the membrane (in both chambers 3 and 5). Thus, the solution onboth sides of the membrane will appear colored showing a positivereaction as in FIG. 4B. At this stage, migration of molecules can behalted by reinserting the non-permeable barrier.

In order for all of the reactions to proceed properly, the correctconcentrations of reagents must be employed. As with other immunoassayprocedures, the concentration of each reagent must be carefullycalibrated based on the expected range of concentrations for the testsamples. Examples of calibration errors and the end result of theseerrors are given in the following paragraphs.

If there is an excess of antibody molecules in chamber 3, then all ormost of the antigens from the test sample will be able to locate bindingsites on the antibody molecules. In this case, few, if any,antigen-chromophore (or antigen-fluorophore) conjugates will bedisplaced; and these conjugates will not migrate to chamber 5 of theapparatus. The end result is that chamber 5 will appear to be relativelycolorless. A test may then be interpreted as negative when it is indeedpositive.

If an excess of antigen-chromogen (or antigen-fluorophore) conjugates isadded to chamber 3 and a small number of antibody molecules are present,then these conjugates may not locate a sufficient number of antibodymolecule binding sites. Thus, the conjugates will be free in solution;and the conjugates will migrate freely through the semipermeablemembrane once it is exposed. This will occur whether or not there isantigen in the test sample. If migration occurs and there is no antigenin the test sample, then color will appear on both sides of the membraneand the reaction will be interpreted as positive when it is indeednegative.

In addition to the aforementioned reaction mechanism for the DITAMassay, alternate reaction mechanisms can be employed in the DITAMapparatus. Any additional alternate reaction mechanisms describedhereinafter may also be employed to fit the needs of the user.

As another example of an exemplary reaction mechanism, anenzyme-substrate system may be used in place of the chromophore orfluorophore. If an enzyme is employed, then an extra step is added tothe reaction. Since the enzyme-antigen conjugate is colorless, anindividual is not able to visualize the presence of this molecule ineither chamber, however, if the appropriate enzyme-substrate solution isadded to chamber 5 of the apparatus, then the enzyme will degrade thesubstrate. For specific enzyme-substrate systems, the degraded substrateis colored. An example of such a system is: horseradish peroxidase asthe enzyme and tetramethylbenzidine plus hydrogen peroxide as the enzymesubstrate. In this case, the degraded substrate has a royal blue color.The intensity of the colored solution is dependent on the concentrationof enzyme. If the reagents are properly calibrated, the color isultimately dependent on the concentration of test sample antigen thatwas able to displace the antigen-enzyme conjugate. Enzyme-substratesystems will be discussed in greater detail hereinafter with anotherembodiment of the DITAM apparatus.

FIGS. 6A-6D and 7A-7C are diagrammatic representations of reactionsequences for another chemical reaction mechanism. In each case, theDITAM apparatus is used in conjunction with chemical vapor detectingequipment. In these reaction sequences, the antibody molecule is labeledwith an enzyme. Labeled antigen is not required. Thus, one less reagentis needed for this assay. The reaction of enzyme with substrate producesvapors which leave the solution and can be detected with the appropriateequipment. These enzyme-substrates may, or may not, produce a colorchange.

The most important criteria in selecting an enzyme-substrate system isto ensure that the resulting products are highly volatile.

FIGS. 6A-6C is a diagrammatic representation of the reaction sequencethat occurs when a test sample contains bacterial or fungal organismsand the DITAM apparatus and assay are to be used in conjunction withchemical vapor-detecting equipment or vapor detection by a user. In FIG.6A, chamber 3 contains antibody molecules (Y-shaped structures in thediagram) 25 that are labeled with enzymes 27 (indicated by an E attachedto each Y-shaped structure). When the test sample is added to chamber 3,bacteria or fungal organisms 29 (large dark spherical structures in thediagram) are introduced into chamber 3. When an individual shakes theapparatus for 1-2 minutes, enzyme-labeled antibodies bind to thebacterial or fungal organisms. This results in the formation of largemolecular weight complexes. Chamber 5 contains an enzyme-substratesolution (the enzyme-substrate molecules 31 are drawn as smalltriangles).

The nonpermeable barrier 9 is removed by lifting upwards, and thisexposes the semipermeable membrane, (FIG. 6B). For this assay, themembrane has a m.w.c.o. of 300,000. Other m.w.c.o. membranes can be useddepending on the type of substance to be tested for. The individualshakes the apparatus in a forward and backward direction for 1 minute.The large molecular weight complexes are not able to migrate through themembrane and these complexes (including the bound enzyme molecules)remain in chamber 3. Thus, little or no enzyme is present in chamber 5to react with the substrate; and volatile substances (vapors) are notproduced for detection through opening 26 in chamber 5.

Any further migration of molecules is halted when the nonpermeablebarrier 9 is replaced (FIG. 6C). Alternately, the enzyme-substratesolution may be added to chamber 5 after the non-permeable barrier 9 hasbeen replaced (FIG. 6C). The cap 7 of chamber 5 is removed andvapor-detecting equipment (not shown) is employed to demonstrate thatvapors are absent. The absence of vapors indicates a positive reaction(specific bacterial or fungal organisms were present in the testsample).

In an alternate reaction sequence to be described hereinafter, a largequantity of vapors indicates a positive test sample and a relativelysmall quantity of vapors indicates a negative test sample.

FIGS. 7A-7C are a diagrammatic representation of the reaction sequencethat occurs when a test sample does not contain bacterial or fungalorganisms and the DITAM apparatus and assay are to be used inconjunction with chemical vapor-detecting equipment or vapor detectionby a user. In FIG. 7A, chamber 3 contains antibody molecules 25(Y-shaped structures in the diagram) that are labeled with enzymes 27(indicated by an E attached to each Y-shaped structure). When the testsample is added to chamber 3, no bacterial or fungal organisms areintroduced into chamber 3. When an individual shakes the apparatus for1-2 minutes, enzyme-labeled antibodies do not have bacterial or fungalcomplexes to bind to; and no large molecular weight complexes areformed. Chamber 5 contains an enzyme-substrate solution (theenzyme-substrate molecules 31 are drawn as small triangles).

The nonpermeable barrier 9 is removed by lifting upwards, and thisexposes the semipermeable membrane (FIG. 7B). For this assay, themembrane has a m.w.c.o. of 300,000. Other m.w.c.o. membranes can be useddepending on the type of substance to be tested for. The individualshakes the apparatus in a forward and backward direction for 1 minute.The antibody-enzyme conjugate has a molecular weight of 218,000 and canmigrate through the membrane. This size is based upon an antibodymolecule with a molecular weight of approximately 150,000 and an enzymemolecule with a molecular weight of approximately 68,000. A variety ofenzyme molecules can be bound to the antibody molecules. The onlyrestriction is that the total molecular weight of the antibody-enzymeconjugate is small enough to pass through the semipermeable membrane.Enzyme-substrate is present in chamber 5 of the apparatus. When theenzyme reacts with the substrate in chamber 5, volatile substances(vapors) 33 are produced.

Any further migration of molecules is halted when the nonpermeablebarrier 9 is replaced (FIG. 7C). The cap 7 of chamber 5 is removed andvapor-detecting equipment (not shown) is employed to demonstrate thatvapors ar present. The presence of vapors 33 indicates a negativereaction (specific bacterial or fungal organisms were not present in thetest 1 sample).

In an alternate reaction sequence to be described hereinafter, a largequantity of vapors indicates a positive test sample and a relativelysmall quantity of vapors indicates a negative test sample.

In another aspect of the invention and with reference to FIG. 8, asecond embodiment of the DITAM assay and apparatus 40 consists of aflexible, cylindrical semipermeable membrane (herein referred to as a"bag" 41) that is tied at both ends 43. The bag 41 is placed in a cappedtest tube 45 or an alternate pocket-sized vial. The size of the vesselcan be varied to suit the needs of the user. For example, a 12 mm×75 mmpolystyrene, round-bottom tube with a cap may be used as the reactionvessel may.

As with the DITAM apparatus described above, a variety of membranes canbe employed; but dialysis-type membranes are the most useful. This isdue to the fact that molecules can easily migrate through the membranewhen an individual is gently shaking the apparatus from side to side. Ifother types of membranes are used, positive or negative pressure may berequired for the molecules to traverse the membrane.

An advantage of the second embodiment of the DITAM apparatus is toincrease the surface area of the membrane to enhance the rate ofmigration through the membrane. Again, all of the directions forperforming the assay can be printed on the reaction vessel 45.

The reagents and supplies needed for performing a DITAM assay using thesecond embodiment are also shown in FIG. 8. The semipermeable membrane41 and test tube 45 have been described above. The following reagentsare placed inside the bag: specific antibody molecules 49,enzyme-labeled antigens 51, and a buffer solution or 0.85% saline. Sincethe membrane must be kept moist and the bag is difficult to reopen,reagents for this assay are not supplied in lyophilized form.

The selection of an enzyme substrate solution depends on the enzyme thatis used to label the antigen. Several different enzyme-substrate systemsmay be employed. An example of one system follows: horseradishperoxidase for the enzyme and tetramethylbenzidine with hydrogenperoxide for the substrate. The vessel cap may be configured to housethe enzyme-substrate solution as indicated by the hatched portion 44 incap 46.

The test sample may contain the antigen (a molecule that is specificallyrecognized by the antibody molecule) or it may be devoid of antigen. Thetest sample may be liquid, i.e., contaminated water, saliva, serum orurine. Alternately, solid test samples, i.e., contaminated dustparticles can be concentrated on cotton swabs and placed in test tubesalong with a buffer solution. Again, these procedures apply to allembodiments of the invention.

FIGS. 9A-9C are diagrammatic representations of the reaction sequencefor performing a DITAM assay using the second embodiment. The individualmay fill the bag 41 with the appropriate test reagents or this bag 41may be pre-filled by a manufacturer. If the bag is prefilled, only thefollowing steps are required for the performance of a DITAM assay: Aliquid test sample is added to a fill line marked on the tube, the tubeis capped, and the individual shakes the tube for approximately twominutes. The enzyme substrate solution is added, and the tube is shakenagain for approximately 1 minute. Then the individual observes the tubefor a color change. The color of the reaction product varies dependingon the enzyme and enzyme substrate utilized.

An absence of color change illustrates a negative reaction. Using theenzyme-substrate described above, a turquoise color illustrates apositive reaction due to the degradation of the substrate(tetramethylbenzidine plus hydrogen peroxide) by the enzyme (horseradishperoxidase). Other colored reaction products are possible depending onthe enzyme-substrate system selected.

FIG. 9A illustrates the bag 41 inside the capped test tube 45. Using thekey in FIG. 8 the bag contains antibody molecules 49 and enzyme-labeledantigens 51 in solution. The antibody molecules 49 are bound to theenzyme-labeled antigens 51. In FIG. 9B, the test sample is added to thetube. This sample is added to a fill line 47 marked on the tube. Whenshaking occurs, the small molecular weight molecules (the antigens 53 inthe test sample) are able to pass through the semipermeable membrane andinto the bag. This event is driven by osmosis due to the difference inthe concentrations of this molecule on each side of the semipermeablemembrane. Once inside the bag 41, the antigens 53 from the test samplecan displace the antigen-enzyme conjugates 51 from the antibodymolecules. A larger concentration of antigen in the test sample willresult in a larger number of displacement reactions. The enzyme-antigenconjugates 51 used for this assay have relatively small molecularweights (compared to the antibody molecules) and are able to migratefrom the inside of the bag 41 to the outside of the bag. Because of thelarge size, the antibody molecules 49 are not able to migrate throughthe membrane and must remain inside the bag. In FIG. 9C, the enzymesubstrate in solution is added to the test tube. Any enzyme moleculesoutside of the bag can degrade the molecules in the substrate solutionrepresented by the black circles 57. This degradation causes a colorchange and indicates a positive reaction by the degrade enzyme-substrate57 as described above. In order to halt the migration of moleculesthrough the membrane, the bag may be removed prior to the addition ofthe enzyme-substrate solution.

As with the DITAM assay shown in FIG. 1A, it is essential that one usesthe correct concentration of assay reagents. Based on the reactionmechanism, an excessive number of antibody molecules inside the bag willessentially hold all or most of the antigen and enzyme-labeled antigenmolecules inside the bag. When filling the bag with reagents, care mustbe exercised not to contaminate the exterior of the bag with any enzymemolecules.

In addition to the aforementioned reaction mechanism for the secondembodiments of the DITAM assay, alternate reaction mechanisms can beemployed and will be described hereinafter.

In another aspect of the invention, a third embodiment of the DITAMapparatus and assay facilitates the detection of large molecular weightsubstances such as proteinaceous antigens from infectious organisms,i.e., bacteria, fungi and parasitic organisms, antibodies directedagainst these organisms, and large molecular weight toxins from theseorganisms. The basic form of the apparatus utilizes a membrane with ahigher m.w.c.o. than the membranes utilized for other versions of theDITAM apparatus. For the third embodiment to be used for the detectionof small molecular weight substances, a membrane with a lower m.w.c.o.is used.

The third embodiment of the DITAM apparatus comprises a tubularsemipermeable membrane 61 (herein referred to as a bag) closed atopposite ends by clamps 63, (FIG. 10A).

The bag 61 (filled with reagents) is placed in a Petri dish 65 or smallvial or other reaction vessel. A wide variety of containers may be usedsuch as a 7 dram snap-cap vial. A 15 dram snap-cap vial (or larger) mayalso be used if larger sample volumes are tested. The snap-cap vesselcan be replaced by a 1 oz. (30 ml) screw-cap vessel or a larger sizescrew-cap vessel. A variety of other types of vessels may be utilized.The limiting factors in selecting any reaction vessel are size, opticalclarity, and lack of reactivity with the reagents used in the assay. Allof the chemical reactions are conducted in this vessel. The vessel mustbe large enough to accommodate the two clamps 63, the semipermeablemembrane 61 and the total volumes of the test sample and theenzyme-substrate solution. The vessel may be configured to enable thestorage of the enzyme-substrate solution in the cap as described above.All of the instructions for the assay can be printed on the side or topof the reaction vessel.

Since the third embodiment of the invention utilizes a somewhat brittleand stiff membrane that must be kept moist, clamps are used to secureboth ends of the membrane. FIG. 10A illustrates the use of Spectra/Por®closures for use with membrane tubing sizes to 15 mm. Spectra/Por®Closures for use with membrane tubing sizes to 35 mm can also be used. Avariety of other types of clamps may be used to seal the membrane atboth ends. Ideally, clamps should only be used once. However, if clampsare to be reused, they must be carefully cleaned to remove any traces ofenzyme. The latter could result in false positive reaction. Heat sealingat both ends of the membrane may also be an option. This would eliminatethe need for clamps.

In this embodiment, the DITAM assay produces a color change in thereaction solution. This can be viewed with the unaided eye. To increaseassay sensitivity, a small portable spectrophotometric device may beused to detect the color change produced by the reaction of enzyme withenzyme-substrate. In another variation of the DITAM assay, the reactionof enzyme with enzyme-substrate produces vapors which can be detectedwith chemical vapor-detecting equipment or a user of the apparatus.

FIGS. 10A and 10B illustrate the use of an antibody-ferritin conjugatefor this embodiment of the DITAM assay. FIG. 10B shows in schematic forman antibody represented by the Y-shaped structure bonded to a ferritinto form the antibody-ferritin conjugate.

FIGS. 11A to 11D illustrate competitive reactions (antigen andenzyme-labelled antigen compete for binding sites on antibodymolecules). The bag 61 has therein an antibody-ferritin conjugate (FIG.11A). The conjugate has a substantially larger molecular weight than theantigens in a test sample. Thus, the conjugate is not able to passthrough the pores of the membrane. This type of reaction is used for thedetection of proteins from bacterial or fungal organisms.

The third embodiment of the DITAM assay can use the following reagents:antibody-ferritin conjugate, antigen-enzyme conjugate, antigen from thetest sample, the enzyme-substrate solution and a buffer solution or0.85% saline (FIGS. 11A-11D). The antibody-ferritin conjugate can bereplaced with any antibody-X conjugate (where X is a large molecularweight substance that prohibits the conjugate from migrating through thepores in the membrane). Examples include: antibody-microspheres andantibody-colloidal gold.

In another variation of the DITAM assay, the following reagents areused: antibody-enzyme conjugate antigen from test sample (where theantigen is a bacterial or fungal organism), the enzyme-substratesolution, and a buffer solution or 0.85% saline. These are illustratedin FIGS. 13A-13C and 15A-15D.

As with the other embodiments of the DITAM assay, reagent concentrationsmust be carefully calibrated. Incorrect concentrations may result intesting errors such as false positives. Also, any contamination of theexterior of the bag or the clamps with enzyme prior to testing willresult in false positive results.

The test sample may be a liquid, i.e., contaminated water, saliva, serumor urine. Alternately, solid test samples, i.e., contaminated dustparticles can be concentrated on cotton swabs and placed in test tubesalong with a buffer solution.

FIGS. 11A-11D and 12 are diagrammatic representations of the reactionsequence for the third embodiment of the DITAM assay. In order toperform this assay, the tubular semipermeable membrane 61 (bag) isclamped at one end. The antibody-ferritin solution 67 is pipetted intothe bag and the bag is clamped at the other end (FIG. 11A). The solutionappears brownish in color due to the ferritin. The bag 61 is then placedin a reaction vessel and covered with a buffer solution or 0.85% salineto keep the membrane moist. Long-term storage may require the additionof an additive to enhance stability and halt or slow the growth of anycontaminants such as bacterial and fungal organisms. Storage at 4° C. isalso recommended if the assay is not performed shortly after filling thebag. To continue with the assay, the bag 61 is removed from the reactionvessel (FIG. 11B). The test sample 69 (which may contain antigen) and asolution of enzyme-labeled antigen 71 are added to the reaction vessel.The bag 61 is then placed in the reaction vessel again, and theindividual shakes the vessel for approximately 2 minutes (FIG. 11C).Afterwards, the bag 61 is removed again. The enzyme-substrate solutionis added to the reaction vessel and the individual observes the vesselfor a color change in the reaction solution (FIG. 11D). Usinghorseradish peroxidase as an enzyme and tetramethylbenzidine withhydrogen peroxide as the enzyme-substrate solution, the positivereaction solution is turquoise or yellow-gold in color. The color andthe intensity of the color are. dependent on the concentration of enzymein the solution. In turn, the concentration of enzyme in the solutioncan be used to determine the initial concentration of antigen in thetest sample. Thus, the greater the color intensity, the greater theconcentration of antigen in the initial test sample. A negative reactionis clear.

Another use of the third embodiment of the DITAM assay uses chemicalvapor-detecting equipment or vapor detection by a user. With referenceto FIGS. 6D, 13A-13C and 14, the tubular semipermeable membrane 61 (bag)is clamped at one end. The antibody-enzyme conjugate 71 (illustrated bya Y-shaped structure with an E attached 27) is pipetted into the bagalong with the test sample. The test sample contains bacteria or fungi(illustrated by dark-circular structures). The bag 61 is clamped at theother end, and it is shaken for 1-2 minutes. To do this, the bag 61 maybe placed in a small container. Alternatively, the bag may be invertedmany times. The bag is then placed in a reaction vessel 65, i.e. a Petridish or other appropriate container, which contains enzyme substratesolution (illustrated by triangular structures 31) (FIG. 13B). Thereaction vessel is shaken for approximately 1 minute. Afterwards, thebag 61 is removed from the reaction vessel (FIG. 13C). Vapor-detectingequipment (not shown) can be used to demonstrate that vapors are absent(a positive test sample as- shown in FIG. 13C). For a negative testsample, vapors would be present.

FIGS. 15A-15D, 16, 17A-17D and 18 also illustrate the DITAM apparatusand assay for use with chemical vapor-detecting equipment. Unlike FIGS.13A-13C and 14, the DITAM assay procedures have been altered slightly sothat the presence of more vapors indicates a positive reaction and lessvapors indicates a negative reaction. This variation permits ease ofinterpreting reaction results. In FIGS. 15A and 17A, the test sample andthe antibody-enzyme conjugates 71 are added to the reaction vessel 65.This vessel is shaken for 1-2 minutes. The bag 61 (a semipermeablemembrane containing buffer and clamped at both ends) is added to thereaction vessel (FIGS. 15B and 17B). After shaking the reaction vesselfor 1 minute, the bag 61 is removed (FIGS. 15C and 17C). Enzymesubstrate solution is added to the reaction vessel, and vapor-detectingequipment is used to determine the amount of vapors present (FIGS. 15Dand 17D).

Using the third embodiment of the DITAM assay, either competitivereactions or displacement reactions may be carried out. For thecompetitive reaction, neither the antigen or the enzyme-labeled antigenare bound to antibody molecules at the beginning of the assay. Both theantigen and the enzyme-labeled antigen are outside the bag at thebeginning of the assay (FIG. 11A). During the shaking step, the antigenand the enzyme labeled antigen can migrate through the pores of themembrane. The antibody-ferritin conjugate is too large to migratethrough the pores of the membrane, and it is retained within the bag 61(FIG. 11C). Both the antigen and the enzyme-labeled antigen haveapproximately equal chances of migrating through the semipermeablemembrane, locating the antibody molecules, and binding to the antibodymolecules. When the bag is removed from the reaction vessel, a portionof the antigen and enzyme-labeled antigens are removed along with thebag. With increasing concentrations of antigen in the test sample, alarger percentage of binding sites on the antibody-ferritin conjugatesare filled by the antigen. Thus, fewer sites are occupied by theenzyme-labeled antigen. These unbound enzyme-labeled antigens can easilymigrate through the pores of the semipermeable membrane. Thus, withincreasing concentrations of antigen in the test sample, theconcentration of enzyme-labeled antigen outside of the bag will alsoincrease. When the bag is removed, there will be larger concentrationsof enzyme-labeled antigen in the vessel to react with the enzymesubstrate solution. The color and intensity of the reaction solution isproportional to the concentration of enzyme-labeled antigen. In turn,this can be related back to the concentration of enzyme-labeled antigen.In turn, this can be related back to the concentration of antigen in thetest sample.

For displacement reactions, e.g. FIGS. 9A-9C, the enzyme-labeled antigenwould be placed in the bag along with the antibody-ferritin conjugate atthe beginning of the assay. These enzyme-labeled antigens are able tobind to the antibody-ferritin molecules in the bag. When the test sampleis placed in the reaction vessel (outside of the bag), the antigens inthe test sample must migrate through the pores of the semipermeablemembrane, locate antibody molecules inside the bag, displace some of theenzyme-labeled antigens from their binding sites on theantibody-ferritin molecules, and finally bind to the antibody-ferritinmolecules. The enzyme-labeled antigens are then free to migrate throughthe pores of the semipermeable membrane to the solution outside of thebag.

The competitive reactions and displacement reactions described above arefor use with enzyme-labeled antigens and enzyme substrates. In bothcases, positive or negative test samples are determined by a change inthe color of the reaction solution.

When using chemical vapor-detecting equipment, the reaction solutiondoes not change color. However, the presence or absence of vapors is anindication of whether or not the test sample was positive or negative.In FIG. 13A, the bag is filled with antibody-enzyme conjugates and thetest sample. The test sample containsbacteria or fungi which bind to theantibody-enzyme conjugates during the shaking step. The bag is thenplaced in a reaction vessel which contains enzyme substrate solution(FIG. 13B). The large complexes are not able to migrate through thepores of the semipermeable membrane. Thus, little, if any, enzyme ispresent in the enzyme-substrate solution when the bag is removed fromthe reaction vessel. Vapors are not produced. When there are nobacterial or fungal organisms in the test sample to bind to theantibody-enzyme conjugate, this conjugate is small enough to passthrough the pores of the semipermeable membrane. When the bag is removedfrom the reaction vessel, antibody-enzyme conjugates are present in theenzyme-substrate solution. When the enzyme reacts with theenzyme-substrate solution, vapors are produced. Vapor-detectingequipment can be used to detect these vapors. For this reactionmechanism, the presence of vapors indicates that the test sample did notcontain specific bacterial or fungal organisms.

The DITAM assay represented in FIGS. 15A-15D, 16, 17A-17D and 18 alsoutilize chemical vapor-detecting equipment. Because of the variation inthe reaction mechanism for these assays, the presence of more vaporsindicates a positive test sample and less vapors indicates a negativetest sample (FIGS. 15A and 17A).

The test sample and the antibody-enzyme conjugates are added to thereaction vessel. During the shaking step, bacterial or fungal organismsbind to antibody-enzyme conjugates to form large complexes. When the bagis added to the reaction vessel and shaken for 1 minute, some of theantibody-enzyme conjugates pass through the pores of the semipermeablemembrane. Large complexes are not able to migrate through the pores ofthe semipermeable due to their large sizes. When the bag is removed fromthe reaction vessel, it either contains buffer or buffer plus a smallquantity of the antibody-enzyme conjugates (FIGS. 15B, 15C, 17B and17C). The enzyme substrate solution is added to the reaction vessel, andvapor-detecting equipment is used to determine the amount of vaporspresent. In FIG. 15D, the large quantity of enzyme in the vessel reactswith the enzyme-substrate to produce volatile substances (vapors). Thelarger amount of vapors indicates that the test sample was positive(specific bacteria or fungal organisms were present in the test sample).In FIG. 17D, the small quantity of enzyme in the vessel reacts with theenzyme-substrate to produce volatile substances (vapors). The smalleramount of vapors indicates that the test sample was negative (nospecific bacteria or fungal organisms were present in the test sample).The appropriate concentrations of reagents must be used for this assay,and the assay must be carefully calibrated in order to determine theamount of vapors that are representative of negative reactions and theamount of vapors that are representative of positive reactions.

Although a primary assay reaction mechanism has been disclosed for eachembodiment of the DITAM apparatus, it should be understood that becauseof the versatility of the invention, many alternate assay reactionmechanism may be employed to suit a user's needs. Thus, the assay hasutility for a greater number of users and for a greater number ofapplications.

As stated above, a wide variety of membranes may be selected for use ineach embodiment of the DITAM apparatus. The selection of membranes isintended to suit the needs of a user. The following provides a listingof several types of membranes that may be used to construct thedifferent versions of the DITAM apparatus. All of these membranes arefrom a single manufacturer, and they are listed here for illustrationpurposes only. Membranes produced by other manufacturers can also beused to construct DITAM apparatuses.

All of the membranes in the following listing are from Spectrum MedicalIndustries, Inc. 60916 Terminal Annex, Los Angeles, Calif., 90060. Themembrane types are provided for the sole purpose of providing examplesof membranes that may be used in the DITAM apparatus. Examples ofmembranes includes (1) SPECTRAPOR® Membrane tubing with any one of thefollowing m.w.c.o.'s: 3,500, 6,000-8,000, 12,000-14,000, (2)Spectra/Por® Membrane tubing with a m.w.c.o. of 50,000, and (3)Spectra/Por® Molecularporous Membrane tubing and Spectra/Por® CEMolecularporous Membrane tubing with any one of the followingm.w.c.o.'s: 25,000, 50,000, 100,000, 300,000, and 500,000. The choice ofm.w.c.o. depends on the type of substance to be tested for. SpectrumMedical Industries, Inc., the manufacturer, describes the Spectra/Por®Molecularporous Membrane and Spectra/Por® CE Molecularporous Membranetubing as nonprotein binding cellulose esters. They are sensitive toorganic solvents, high temperatures, and should not be allowed to dry.SPECTRAPOR® Membrane tubing type (1) above is supplied by themanufacturer in a "dry" form while types (2) and (3) above are suppliedby the manufacturer is "moist" form (in a 0.05% sodium azide solution).For these membranes, moisture is a critical factor in performance. Thus,the membrane must be kept moist in order to ensure optimal performancein the DITAM apparatus and assays. For prolonged periods of time, themembranes in the DITAM apparatus can be kept "wet" in a 0.05% sodiumazide solution until the DITAM apparatus are employed. At that time, thesolutions may be discarded and replaced with the test solution. If thepresence of 0.05% sodium azide interferes with the DITAM assay, then theinterior of the apparatus may be washed with a buffer solution or 0.85%saline solution prior to use.

The aforementioned membranes are available from the manufacturer indifferent flat widths and cylinder diameters. Membranes with a flatwidth of 12 mm (cylinder diameter of 7.5 mm) and a flat width of 34 mm(cylinder diameter of 21.6 mm) may be employed in the DITAM apparatus. Avariety of sizes can be employed to suit the needs of the end user.

Additional equipment may be required to increase the sensitivity of theassay or to give a quantitative result rather than a qualitative resultfor the assay. Optical density detectors, small spectrophotometers, andchemical-vapor detecting equipment have been mentioned previously. Thetype of detector will depend on the type of reagents and reactions foreach DITAM assay. Since these detectors are well known in the art,further description is not provided.

Although reagents useful for performing the various DITAM assays havebeen disclosed above, other reagents may serve as alternatives. Antigensmay be labeled with chromophores, fluorophores, enzymes that producecolored compounds upon reaction with the appropriate substrates, enzymesthat produce highly volatile substances (vapors) upon reaction with theappropriate substrates, iron-containing molecules, radioactivesubstances, or any other molecule that can be bound to the antigen andutilized as a signal that a reaction has occurred.

Either monoclonal or polyclonal antibodies can be used for the DITAMassay. If a single substance is to be detected, then antibodies with asingle specificity would be selected. However, if multiple substancesmust be screened for in a single DITAM assay, then several differentbatches of antibodies (each having a different specificity) would beselected. When testing for small molecular weight substances, antibodiesof the IgG Class may be utilized without further alteration. Whentesting for large molecular weight substances, antibodies of the IgGclass can be conjugated to ferritin or to microspheres or to colloidalgold (FIG. 19) or a wide variety of other large molecules. Thesemolecules should not restrict the activity of the antibody molecules andthey should have a size that is large enough to prohibit theantibody-large molecule complex from migrating through the pores in theDITAM apparatus. Alternatively, antibodies of the IgM Class may beemployed since they are substantially larger in size than antibodies ofthe IgG Class.

For DITAM assays requiring chemical vapor-detection equipment, theantibodies can be labeled with any of a wide variety of enzymes thatwill react with the appropriate substrate to produce volatile substance(vapors). The selection of enzyme depends on the type of vapor-detectionequipment and the needs of the end user.

Receptor molecules may be used as a replacement for antibody moleculesin any of the DITAM assays. In either instance, the recognition moleculemust be capable of binding to the specific substance of interest(analyte) in the test sample. In addition, the type of chemical bondmust be weak enough to enable association and disassociation reactionsto occur. Thus, an analyte in a test sample can displace alabeled-analyte that is bound to the receptor molecule. Unbound analytemolecules (either labeled or unlabeled) can easily pass through thepores of the semipermeable membrane (bag).

For the various embodiments of the DITAM apparatus and assays,directions for performing each type of assay have been disclosed. Due tothe versatility of the DITAM assay, other variations in assay proceduresare possible. Examples of two of these variations follows. Both involvecompetitive-type antigen-antibody reactions. The first variation is forthe detection of small molecular weight substances, and the secondvariation is for the detection of large molecular weight substances.

In order to detect small molecular weight substances, an individualplaces a known quantity of specific antibodies and enzyme-labeledantigens in the reaction container. The test sample, which contains anunknown quantity of antigen, is also placed in the reaction vessel. Theenzyme may be substituted with another molecule as described above. Thecontainer size depends on the use's needs. The vessel is shaken forapproximately 2 minutes. A bag (as described previously) is placed inthe reaction vessel. The molecular weight cutoff of the bag will dependon the substance to be tested for. The bag contains either a buffersolution or 0.85% saline. Alternatively, the bag contains the same typeof specific antibody molecules that have been added to the solution inthe reaction vessel. The reaction vessel is shaken for approximately 1-2minutes. Then the bag is removed. Enzyme substrate is added to thereaction vessel. The individual observes the color of the solution inthe reaction vessel. Ideally, a small spectrophotometer should be usedto product a quantitative result. In addition to the test sample,negative and positive control samples should be tested for comparisonpurposes. A decrease in color and intensity in the reaction solutionsindicates an increase in the amount of antigen in the test sample. Thus,the color of the control solutions should be compared to the color ofthe test solution.

In order to detect large molecular weight substances, an individualplaces a known quantity of specific antibody molecules (bound to largemolecular weight substances such as ferritin) and enzyme-labeledantigens in the reaction vessel. The test sample, which contains anunknown quantity of antigen, is also placed in the reaction vessel. Theenzyme may be substituted with another molecule as described above. Thecontainer size depends on the user's needs. The vessel is shaken forapproximately 2 minutes. A bag (as described previously) is placed inthe reaction vessel. The molecular weight cutoff of the bag will dependon the substance to be tested for. The bag contains either a buffersolution or 0.85% saline. Alternatively, the bag contains the same typeof specific antibody molecules (bound to large molecular weightsubstances such as ferritin) that have been added to the solution in thereaction vessel. The reaction vessel is shaken for approximately 1-2minutes. Then the bag is removed. Enzyme substrate is added to thereaction vessel. The individual observes the color of the solution inthe reaction vessel. Ideally, a small spectrophotometer should be usedto produce a quantitative result. In addition to the test sample,negative and positive control samples must be tested for comparisonpurposes. A decrease in color and intensity in the reaction solutionsindicates an increase in the amount of antigen in the test sample. Thus,the color of the control solutions should be compared to the color ofthe test solution.

For both of the aforementioned variations in the DITAM assay, antigenmolecules from the test sample compete with enzyme-labeled antigens forbinding sites on the specific antibody molecules. This occurs during thefirst shaking step. With increasing concentrations of antigen in thetest sample, more binding sites on the antibodies will be filled bythese antigens; and fewer binding sites on the antibody molecules willbe filled by the enzyme-labeled antigens. When the bag (semipermeablemembrane) is added to the reaction vessel, unbound antigen and unboundenzyme-labeled antigen molecules freely pass through the pores of themembrane. This occurs during the second shaking step. The antibodymolecules or antibody molecules bound to large molecular weightsubstances are not able to pass through the membrane due to their sizes.When the bag is removed from the reaction vessel, the antigens andenzyme-labeled antigens inside the bag are also removed. With theremoval of a substantial number of enzyme-labeled antigen molecules whenthe bag is removed, there will be fewer enzyme-labeled antigen moleculesin the reaction vessel to react with the enzyme-substrate solution andproduce a color change. Thus, with increasing concentrations of antigenin the test sample, the color of the reaction solution will decrease inintensity.

When specific antibody molecules or specific antibody molecules (boundto large molecular weight substances such as ferritin) are containedwith the bag, antigens and enzyme-labeled antigens will bind to theseantibodies. This may prevent or slow down the rate of migration of thesesmall molecules from the inside to the outside of the bag. Thus, betterresults could be achieved.

As such, an invention has been disclosed in terms of preferredembodiments thereof which fulfill each and every one of the objects ofthe present invention as set forth hereinabove and provides an improvedassaying apparatus and method for detecting a wide range of molecularweight substances.

Of course, various changes, modifications and alternations from theteachings of the present invention may be contemplated by those skilledin the art without departing from the intended spirit and scope thereof.Accordingly, it is intended that the present invention only be limitedby the terms of the appended claims.

I claim:
 1. A rapid method for competitive displacement immunoassayscharacterized by diffusion through a membrane to separate free taggedanalyte from bound tagged analyte to detect an analyte in the form ofantigens contained in substances selected from the group consisting ofhazardous wastes, pesticides, toxic chemicals, chemical warfare agentsand infectious organisms, said method comprising the steps of:(1)assembling a detection device having (A) reagents for performing saidimmunoassay comprising tagged analyte-antibody complexes and (B) amulti-chambered container having a semi-permeable membrane securedbetween chambers, the semi-permeable membrane having a predeterminedmolecular weight cut off to allow diffusion between chambers of analyteand tagged analyte and to prevent passage between chambers ofanalyte-antibody complexes and tagged analyte-antibody complexes, saidreagents having predetermined analyte binding characteristics to effectindirect detection of the analyte, said reagents being distributed inone or more alternating chambers, said adjacent chambers having aremovable nonpermeable barrier adjacent to the semipermeable membranewherein fluid communication between adjacent chambers is controlled byremoval or insertion of said nonpermeable barrier, (2) processing asample of suspected analyte in the detection device by measuring for thepresence or absence of free tagged analyte and correlating the taggedanalyte with the predetermined binding characteristics of the reagents,and (3) determining whether the analyte is present.
 2. The method ofclaim 1 wherein the tagged analyte-antibody complexes are tagged with achromogen or a fluorophore.
 3. The method of claim 2 wherein the taggedanalyte antibody complexes are tagged with a chromogen.
 4. The method ofclaim 2 wherein the tagged analyte-antibody complexes are tagged with afluorophore.
 5. The method of claim 1 wherein the molecular weight cutoff is in the range of 3,500 to 500,000 daltons.